Viral fusion proteins are dynamic fusion machines that drive membrane fusion by irreversible protein refolding from a metastable pre-fusion conformation to a stable post-fusion conformation. The fusogenicity of the protein is important for viral infection.
The fusion proteins of enveloped viruses can be classified in different types based on the general irreversible folding mechanism they display to drive fusion of the virus with the target cell. Fusion proteins from unrelated viruses, such as the fusion protein F from Paramyxoviridae, Ebola GP, Retroviridae envelope protein, Coronaviridae spike, Herpesviridea gB, Orthomyxovirideae Hemagglutinin (HA) and Hemagglutinin Esterase (HE), and others are classified as class I fusion proteins and refold from a labile pre-fusion state to a stable post-fusion state through a similar mechanism although they do not exhibit any significant sequence homologies. Class I fusion proteins thus fuse the viral and host-cell membranes by irreversible protein refolding from the labile pre-fusion conformation to the stable post-fusion conformation. Structures have been determined for a variety of class I fusion proteins in pre-fusion conformation and post-fusion conformation providing insight into the mechanism of this complex fusion machine.
Except for Ebola GP and herpes gB, typically, the inactive mature class I fusion protein (e.g. F0 for paramyxoviruses, HA for Orthomyxoviruses) requires cleavage during intracellular maturation, often by a furin-like protease that results in an N-terminal part and an C-terminal part. The cleavage site is near or adjacent to a stretch of 20-25 hydrophobic amino acids (the fusion peptide), followed by a heptad repeat region in the C-terminal part. Since these are class I membrane proteins, the C-terminus contains the transmembrane domain (TM) and after cleavage the membrane bound C-terminal part exposes the N-terminal hydrophobic fusion peptide (FP) (FIG. 1). In order to refold from the pre-fusion to the post-fusion conformation, there are two regions that need to refold, which are referred to as the refolding region 1 (RR1) and refolding region 2 (RR2). For all class I fusion proteins, the RR1 includes the FP and heptad repeat A (HRA). After a trigger, the HRA's of all three protomers in the trimer transform from a helix, or from an assembly of loops and secondary structures, to a long continuous trimeric helical coiled coil (FIG. 1). The FP, located at the N-terminal segment of RR1, is then able to extend away from the viral membrane and inserts in the proximal membrane of the target cell. Next, the refolding region 2 (RR2), which is located C-terminal to RR2 closer to the TM and often includes the heptad repeat B (HRB), relocates to the other side of the fusion protein and binds the HRA coiled-coil trimer with the HRB domain to form the six-helix bundle (6HB) or with an extended polypeptide chain like Influenza HA. These similarities have been recognized by a nomenclature that places viral fusion proteins with these sequence and structural features into the so-called class I viral fusion protein group (Earp et al. Current topics in microbiology and immunology 185: 26-66, (2005); Jardetzky et al. Current opinion in virology 5: 24-33 (2014)).
When viral fusion proteins are used as a vaccine component the fusogenic function is not important. In fact, only the mimicry of the component is important to induce cross reactive antibodies that can bind the virus. Therefore, for development of robust efficacious vaccine components it is desirable that the meta-stable fusion proteins are maintained in their pre-fusion conformation. A stabilized fusion protein in the pre-fusion conformation can induce an efficacious immune response.